CN109021078B - Dendritic cell-targeting affinity peptide TY peptide and application thereof - Google Patents

Abstract

The application belongs to the technical field of protein correlation for tumor therapy, and particularly relates to an affinity peptide TY peptide targeting dendritic cells and application thereof. The TY peptide is a 12 peptide, and the specific amino acid sequence is as follows: TITHFQFGPTVY are provided. On the basis of TY peptide, a novel nano transportation system capable of targeting DC is further designed on the basis of MSN, and a mode antigen OVA and an immune adjuvant CpG are loaded, so that a nano carrier MSN-TY/OVA/CpG is successfully prepared. Experiments show that: MSN-TY/OVA/CpG can be effectively absorbed by immature DC, thus improving the efficiency of antigen absorption by DC, promoting the maturation of DC, effectively activating DC, effectively promoting the processing and presentation of antigen, stimulating the secretion of cell factor, and further triggering antigen-specific CTL immune response and anti-tumor immune response.

Description

Dendritic cell-targeting affinity peptide TY peptide and application thereof

Technical Field

The application belongs to the technical field of protein correlation for tumor therapy, and particularly relates to an affinity peptide TY peptide targeting dendritic cells and application thereof.

Background

Tumors seriously threaten human health, and tens of millions of new cases and death cases exist in the world every year, wherein China accounts for about one fourth. Surgical treatment, radiotherapy and chemotherapy are currently the conventional protocols for the clinical treatment of tumors. Among the major problems facing tumor therapy are metastasis and high recurrence rate of late stage tumors; chemotherapy and radiotherapy have large toxic and side effects, and the treated tumor is easy to generate resistance. Therefore, the development of highly effective, highly specific and low-toxic drugs is a urgent necessity for the current tumor therapy.

The tumor immunotherapy makes full use of the activation mechanism of the body's own immune system to eliminate tumor cells, and is a major breakthrough in the history of tumor therapy. The currently used forms of tumor immunotherapy are mainly cytokine therapy, blocking immune checkpoint therapy, cellular immunotherapy, especially chimeric antigen receptor T cell immunotherapy (CAR-T), and tumor vaccine therapy, and these therapeutic strategies have also achieved clinically favorable results. But also suffer from problems such as high cost, a large variation in individual immune response during treatment, and negative reactions (such as complications of cytokine release). The tumor vaccine has the unique advantages of being capable of loading tumor-related antigens and exciting antigen-specific anti-tumor immune response, and the like, and becomes a hotspot of the current research.

The tumor vaccine can promote antigen-specific CD8⁺Proliferation of T cells stimulates activation of cytotoxic T lymphocytes and thus effective elimination of tumor cells. Antigen presenting cells play an important role in T cell mediated immune responses. At present, DC cells are the best known professional antigen presenting cells with the strongest functions and the only ability to activate resting state T cells, are core components for starting, maintaining and regulating immune response, and play a key role in tumor immunotherapy.

A plurality of potential targets exist on the surface of the DC, and the research of improving the antigen transportation efficiency by coupling the antigen to the antibody by a chemical method or fusing the antigen and the DC-targeted antibody from a gene level provides a new strategy for the development of tumor vaccines at present. The antibody mediates the endocytosis of the antigen, improves the transport efficiency of the antigen, and activates the immune reaction mediated by the T cell. But also face problems, such as antibodies may elicit a negative immune response in vivo, which in turn affects T cell activation; the antigen-antibody complex has poor tissue permeability, and the antibody of murine origin can produce high immunogenicity when applied to human. Therefore, the discovery of potential targeting molecules on the DC surface, the targeting transportation of antigens and the promotion of the cross presentation of exogenous antigens are very critical to the stimulation of CTL immune response and are hot spots of the current tumor vaccine research. In the prior art, tumor vaccines based on polypeptide-targeted DC surface specific receptors have been applied to tumor immunotherapy, which can effectively activate immune response in vivo, thereby resisting tumor invasion. However, the primary application shows that the existing tumor vaccine has the defect of weak capability of stimulating anti-tumor immune response, and the main reasons are that the antigen transportation efficiency is low, the in vivo degradation rate is high, the antigen cross presentation capability is weak, most of the antigen cross presentation capability is identified by non-professional antigen presenting cells to cause the antigen off-target effect, and then the incapability of CTL is induced, so that the effect of treating the tumor is weakened, therefore, a new targeted DC molecule is screened and a novel tumor vaccine is designed, and the method has important significance for improving the treatment effect of the tumor vaccine.

Disclosure of Invention

The application aims to provide an affinity peptide TY peptide targeting Dendritic Cells (DC), and an MSN-TY/OVA/CpG nano-carrier is prepared aiming at the affinity peptide, so that a foundation is laid for treating related diseases.

The technical solution adopted in the present application is detailed as follows.

An affinity peptide TY peptide targeting dendritic cells is a 12 peptide (containing 12 amino acids), the amino acid sequence of the peptide is shown as SEQ ID NO.1, and the specific amino acid sequence is as follows: Thr-lle-Thr-His-Phe-Gln-Phe-Gly-Pro-Thr-Val-Tyr, i.e., T-I-T-H-F-Q-F-G-P-T-V-Y (TITHFQFGGPTVY).

The application of the affinity peptide TY peptide of the targeted dendritic cell in preparing an anti-tumor vaccine.

The preparation method of the affinity peptide TY peptide of the targeted dendritic cell is an Fmoc solid phase synthesis method.

The nano-carrier MSN-TY prepared by the affinity peptide TY peptide targeting dendritic cells is prepared by acetylating TY peptide and then covalently coupling the acetylated TY peptide to the surface of MSN (mesoporous silica), and the specific construction process is as follows:

(1) first, an acetylated TY peptide (or an acetylated TY peptide prepared directly by Fmoc solid phase synthesis) was prepared,

(2) then, dissolving acetylated TY peptide in 2 mL of DMF, adding 10-fold equivalent of HoBt for mixed incubation, then adding 10-fold equivalent of DIC, uniformly mixing with the reactant, and incubating at room temperature for 10 min to activate carboxyl on the TY peptide;

(3) according to MSN (amination modification): measuring an appropriate amount of MSN (polypeptide =1: 1) (mass ratio) in a clean 10 mL round-bottom flask, and redispersing the MSN in 2 mL of DMF by using an ultrasonic cleaner;

(4) adding the activated TY peptide in the step (2) into the MSN subjected to ultrasonic dispersion in the step (3), and carrying out magnetic stirring reaction for 24 hours at room temperature; the reaction product was washed 3 times with water to remove unbound polypeptide, and then dispersed in PBS buffer (pH = 7.2), and the above reactions were all performed with exclusion of light.

Firstly, acetylating TY peptide, covalently coupling the acetylated TY peptide to the surface of MSN (mesoporous silica) (MSN-TY), and then adsorbing a mode antigen OVA protein and a TLR9 receptor agonist CpG to the surface of MSN-TY by an electrostatic adsorption method, thereby finally preparing the nano vaccine carrier MSN-TY/OVA/CpG capable of specifically targeting DC, wherein the TY peptide can enable the carrier to specifically target DC, and the loaded mode antigen OVA protein and the TLR9 receptor agonist CpG can efficiently activate DC; the nano-carrier has regular morphology and average particle size of about 150 nm.

The nano-carrier MSN-TY/OVA/CpG is applied to the preparation of anti-tumor vaccines.

In the application, the inventor firstly utilizes the phage display peptide library technology to screen and obtain the affinity peptide TY peptide targeting the mouse spleen DC, further synthesizes the DC affinity peptide labeled by biotin through the Fmoc solid phase synthesis technology, and utilizes an in vitro affinity experiment and a flow detection method to prove that the TY peptide obtained by screening has better affinity.

Development of anti-tumor vaccines based on Dendritic Cells (DCs) is a hotspot of current immunotherapeutic studies by promoting antigen-specific CD8⁺Proliferation of T cells stimulates activation of cytotoxic T lymphocytes, thereby effectively eliminating tumor cells. There are many potential targets on the DC surface, and targeting DC trafficking antigens is a potential method for preparing tumor vaccines. However, the main problems of the existing tumor vaccines are antigen off-target effect (off-target effect) and the inability to efficiently stimulate the body to generate anti-tumor immune response. Therefore, the selection of DC surface specific receptor, the design of novel tumor vaccine, the activation of T cells from multiple links, and the important significance for the immunotherapy of tumor. Based on the aim, the application successfully prepares the specific DC-targeting nano-carrier MSN-TY/OVA/CpG, the carrier has uniform particle size and high spleen DC uptake efficiency, and can stimulate DC maturation, trigger antigen cross presentation and obviously stimulate T cell proliferation. The vector provides new thought and technical support for developing new anti-tumor vaccines.

In general, on the basis of obtaining the specific targeting DC polypeptide TY peptide by screening, a novel nano transportation system capable of targeting DC is further designed on the basis of MSN, and a mode antigen OVA and an immune adjuvant CpG are loaded, so that a nano carrier MSN-TY/OVA/CpG is successfully prepared. Further in vitro and in vivo experimental effect evaluation shows that: MSN-TY/OVA/CpG can be effectively taken up by immature DC, thus improving the efficiency of antigen taking by DC, promoting the maturation of DC, effectively activating DC, effectively promoting the processing and presentation of antigen, stimulating the secretion of cell factors, further triggering antigen-specific CTL immune response and anti-tumor immune response, and effectively stimulating antigen-specific CD8 in a B16-OVA tumor-bearing model⁺T cell immune response, show higher improvement effect.

Drawings

FIG. 1 shows the case of flow detection of phage monoclonal affinity DCs;

FIG. 2, FIG. 3 and FIG. 4 are mass spectra and OVA of biotinylated TY peptide, respectively_257-264Peptide mass spectrum, acetylated TY peptide mass spectrum;

FIG. 5 is an affinity assay for DC affinity peptide and immature BMDC with GA peptide as a control peptide; the results show that only TY peptide can be attached to BMDC;

FIG. 6 is a graph of MSN-TY preparation, in which (A) the IR spectrum analysis of MSN, MSN-TY and MSN-TY/OVA/CpG; (B) thermogravimetric analysis plots of MSN and MSN-peptide;

FIG. 7 shows MSN-TY/OVA/CpG vector preparation; (A) BCA quantifies the amount of free protein in the supernatant, and the encapsulation rate of the MSN loaded OVA protein under different proportions is calculated; (B) detecting the condition of loading CpG on the MSN by agarose gel electrophoresis;

FIG. 8 shows the characterization of nanocarriers, (A) TEM images of different nanocarriers, (B) images of MSN dispersed in water, (C) SEM images of MSN, (D) Zeta potentials of MSN, OVA, MSN-TY/OVA/CpG; (E) the particle size distribution of different nano-carriers;

FIG. 9 shows cytotoxicity and uptake assays, (A) OVA/CpG, MSN/OVA/CpG, MSN-TY/OVA/CpG nanocarriers were incubated with BMDC cells for 48 h, and their cytotoxicity was tested using MTT assay; (B) OVA_FITC/CpG、MSN/OVA_FITCCpG and MSN-TY/OVA_FITCCpGs (with OVA for each group)_FITCAmount of 20. mu.g/mL) was incubated with spleen cells at 37 ℃ for 4 hours and flow-detection results were obtained; (C) a statistical value of fluorescence intensity; (D) BMDC respectively with OVA_FITCAfter incubation for 4h with different nano-carriers, nucleus (blue) and lysosome (red) are respectively stained by Hoechst and Lysotracker, and OVA (OVA) uptake of BMDC is observed by a laser confocal microscope_FITCAnd in the case of nanocarriers;

FIG. 10 is a graph of the effect of OVA/CpG, MSN/OVA/CpG, and MSN-TY/OVA/CpG nanocarriers on DC maturation; different nano-carriers and 2 x 10⁶Individuals/wells were incubated in 6-well plates at 37 ℃ for 24h (the amount of vector added per well was calculated as OVA 100. mu.g and CpG 80. mu.g) to flow-test for changes in the DC surface co-stimulatory molecules CD86 (A, C) and CD40 (B, D); (E, F) ELISA detection is carried out to respectively detect the secretion amount of the cytokines TNF-alpha and IL-12p70 in the culture medium supernatant;

FIG. 11 is the ability of nanocarriers to cross-present antigens in vitro; OVA/CpG, MSN/OVA/CpG and MSN-TY/OVA/CpG, PBS group as control group (final concentration of 50. mu.g/mL calculated according to the amount of OVA contained) was incubated with BMDC (Day 6) at 37 ℃ for 4h, washed twice with serum-free medium, and then with CD8 selected from OT-I mice⁺T cells (CFSE labeled), BMDCs at the following ratio: CD8⁺T =1: 10 are paved in a 96-well plate and incubated for 72 h at 37 ℃; (A) flow detection of CD8⁺Proliferation of T cells; (B) detecting the secretion amount of IFN-gamma in the supernatant by ELISA; (C, D) qRT-PCR detecting the relative expression of Perforin and Grz B on the mNAN level;

FIG. 12 shows antigen-specific CD8 elicited by OVA/CpG and various nanocarriers⁺A T cell immune response; (A) a protocol for immunizing a mouse; (B) spleen cells were stimulated with 10. mu.g/mL OVA257-264 antigen peptide for 6h in vitro, and staining with intracellular factor detects CD8⁺The expression level of IFN-gamma in T cells, wherein the left graph is a flow representation graph of different treatment groups, and the right graph is a statistical graph; (C) OVA for spleen cells_257-264Stimulating in vitro for 5 days, and detecting the secretion of IFN-gamma in the supernatant by ELISA; (D, E) qRT-PCR detecting the relative expression of perforin and GrzB;

FIG. 13 is a graph of the anti-tumor effect of MSN-TY/OVA/CpG on the B16-OVA model; (A) body weight change profile of mice during administration of the nanocarrier immunotherapy regimen (B); (C) tumor volume change curves for mice; (D) fifth day after the last dose, mice were sacrificed, photographs of the tumors of the mice;

FIG. 14, FIG. 15, FIG. 16 shows MSN-TY/OVA/CpG anti-tumor effects; (A) tumor site CD3⁺CD8⁺Infiltration of T cells; (B-E) mice dLN and spleen Using OVA_257-264Re-stimulating in vitro for 6h, and respectively detecting IFN-gamma in dLN by flow⁺CD8⁺、perforin⁺CD8⁺And GrzB⁺CD8⁺And IFN-gamma in spleen⁺CD8⁺The ratio of (A) to (B); OVA for (F, G) dLN and spleen cells_257-264Stimulating in vitro for 5 days again, and detecting the secretion of IFN-gamma in the supernatant by ELISA; (H, I) relative Table of the detection of perforin and GrzB by qRT-PCRAnd (4) obtaining the amount.

Detailed Description

The present application is further explained below with reference to examples, and before describing specific examples, the following examples are briefly described with reference to experimental background conditions such as experimental materials.

Experimental animals: female C57BL/6J mice (SPF grade), 6-8 weeks old, purchased from Experimental animals technology, Inc. of Weitongli, Beijing; OT-I mice, offered by Ministry of Yang Kangchong Ming and Ming, Shanghai university of transportation;

the main reagents are as follows:

Ph.D-12TM phage display peptide library kits, product of NEB; EasySep Mouse CD11c Positive Selection Kit II (for separating and sorting CD11c cells), product of Stemcell; DNase I, beta-propiolactone, Ovalbumin (OVA), Sigma-Aldrich; collagenase IV, product of Invitrogen corporation; biotinylated anti-M13 antibody, Bioengineered bioengineering, Inc.; Streptavidin-PE antibody, product of eBioscience; CpG ODN1826, jinzhi biotechnology limited; BCA protein quantification kit, Beijing prilley Gene technology, Inc.; MSN (aminated modification), shanghai carboxyphenanthrene biotechnology; amino acids, Biotin-lysine, Rink resin, Wang resin and the like used in the solid phase synthesis of Fomc are all purchased from Shanghai Jier Biochemical Co., Ltd;

other materials such as LB medium, top agar (0.5 g yeast powder, 0.5g NaCl and 1g tryptone, 0.1g MgCl 2.6H2O, 0.7 g agarose, 100 mL fixed with double distilled water), X-gal/IPTG solution, LB plate containing IPTG/X-gal, glycine-hydrochloric acid eluent (Gly-HCl, 0.2M, pH = 2.2), Tris-HCl neutralizing solution (pH = 9.1), PBS Buffer, PEG/NaCl solution (20 g molecular weight 8000 PEG, 14.61 g NaCl, 100 mL fixed with double distilled water), EasySepTM Buffer (PBS containing 1 mM EDTA and 2% FBS (v/v), pH 7.2), etc. can be prepared conventionally according to the prior art;

the main apparatus is as follows:

FACS Calibur flow cytometer, BD corporation; polypeptide synthesizer, Jian bang pharmaceutical Co, Hainan; RP-HPLC purification System, Shimadzu, Japan; transmission Electron microscopy (Tecnai G2F 20-S-TWIN), FEI Inc.; scanning electron microscope (Sigma 500), Carl Zeiss; infrared spectrometer (FT/IR), Thermoscientific, Inc.

Example 1

The affinity peptide TY peptide of the targeted dendritic cell is obtained by screening through a phage display peptide library technology, the screening process comprises the steps of sorting mouse spleen DC cells, panning at a cell level, amplifying phage, determining monoclonal DNA sequence of affinity DC phage and the like, and the specific screening process is briefly introduced in the embodiment as follows.

(I) sorting mouse spleen DC cells

Selecting C57BL/6J mice with 6-8 weeks, killing and disinfecting (soaking in 75% alcohol for about 5 min), and then preparing DC cells according to the prior art, or specifically referring to the following operations:

(1) dissecting the mouse, taking out spleen, cutting into pieces, adding collagenase I and DNA IV enzyme to digest spleen cells, and shaking and digesting for 30 min at 37 ℃ in a shaking table;

(2) grinding splenocytes with glass slide, filtering with 70 mesh filter screen to obtain single cell suspension, adding 10 mL PBS 7.2, centrifuging at 4 deg.C and 2000 rpm for 5 min, and discarding supernatant; adding 5mL of erythrocyte lysate, gently blowing and beating uniformly by using a pipette gun, performing room-temperature lysis for 8 min, and washing twice by using 7.2 sterile PBS (phosphate buffer solution); adding appropriate amount of EasySepTM Buffer to resuspend cells, adjusting density to 1 × 10⁸Per mL, cells are transferred to a special sorting tube;

(3) sealing the rat serum volume of 50 muL according to 1 mL of cell suspension at room temperature for 3 min; placing 25 muL of each of the antibody component A and the antibody component B in a low-adsorption 1.5mL EP tube, uniformly mixing, incubating the antibody component A and the antibody component B at room temperature for 5 min, adding the mixture (50 muL/mL) of the antibody component A and the antibody component B into the sealed cell suspension, uniformly blowing by using a gun, and incubating at room temperature for 5 min;

(4) violently shaking and uniformly mixing streptavidin magnetic beads in a Kit (EasySep Mouse CD11c Positive Selection Kit II) on a vortex instrument for 30 s to ensure that the beads are uniformly dispersed; adding the uniformly mixed magnetic beads into the cell suspension according to the amount of 40 muL/mL, and incubating for 3 min at room temperature after uniformly blowing; adding a proper amount of EasySepTM Buffer into the cell suspension until the final volume is 2.5 mL, and gently blowing, beating and uniformly mixing;

(5) placing the sorting tube in a special magnet of the kit, and standing at room temperature to enable the sorting tube to be adsorbed for 2.5 min; and (4) taking up the magnet, continuously suspending and dumping the liquid in the sorting tube into a new centrifugal tube, wherein the cells adsorbed on the tube wall are the DC cells obtained by positive sorting, further adding PBS (phosphate buffer solution) 7.2 for heavy suspension, and counting for later use.

(II) cell horizontal panning process

The cell level panning is carried out by referring to the specification of the phage surface display peptide library, and the specific operation process can be referred to as follows.

And adding the sorted DC cells into M13 empty phage inactivated by beta-propiolactone (empty phage without random 12 peptide inserted) for sealing, then adding a phage propeptide library, and incubating for 1h at 4 ℃ with mild shaking. PBST (PBS = 5.0) was added, washed 5 times, and further washed 5 times with PBST (PBS = 7.4). Adding into proper amount of sterile water, and lysing cells at room temperature while continuously blowing. Eluting with glycine elution buffer solution (pH 2.2), centrifuging, transferring the supernatant to a new EP tube, adding a neutralizing solution for neutralization, and amplifying and purifying the eluted phage by using E.coli ER2738 to obtain the peptide library of the next round. During the screening process, the above procedure was repeated to start the next round of affinity screening.

In the implementation process of this example, four rounds of screening were performed to obtain phage single clones with specific affinity to DC. And respectively measuring the titer of the phage after elution and amplification, calculating the recovery rate of each round according to a formula, wherein the recovery rate = the total number of the eluted phage/the total number of the input phage multiplied by 100%, and evaluating whether the phage is enriched according to the recovery rate.

After the above four rounds of binding-elution-amplification series screening, the phage recovery rate of each round is counted as shown in the following table:

。

from the recovery results in the table above: through four rounds of panning, the phage with affinity to DC is enriched, the recovery rate is gradually increased, and sequencing of phage insertion DNA sequence can be carried out, so as to further screen out monoclonal with good binding effect.

Based on the screening results (44 in total) of the isolated blue plaques on the fourth round of plate, sequencing analysis is further carried out after culture to obtain the DNA sequence inserted in the phage, and further obtain the polypeptide sequence of the specific affinity DC cell.

After sequencing analysis, 8 peptide sequences (DK, TY, SL, QM, SP, NK, ER, EL) with better affinity with DC are obtained, wherein TY peptide is the polypeptide claimed by the application (other polypeptide sequences are not separately illustrated because of different polypeptide sequences from the polypeptide sequence claimed by the application).

The sequencing result shows that: the TY peptide is a 12 peptide (containing 12 amino acids), and the specific amino acid sequence is as follows: Thr-lle-Thr-His-Phe-Gln-Phe-Gly-Pro-Thr-Val-Tyr, i.e., T-I-T-H-F-Q-F-G-P-T-V-Y (TITHFQFGGPTVY).

Furthermore, the affinity of the TY phage monoclonal inserted with TY peptide and DC is preliminarily identified by phage affinity experiment, and the specific experimental process is as follows:

firstly, amplifying screened target phage monoclone; next, the cell density of the single cell suspension of mouse spleen cells was adjusted to 1X 10 with sterile PBS 7.2⁷Each tube is divided into 100 mu L (1 multiplied by 10) per mL⁶Per tube); finally, each tube was added with the corresponding amplified phage (10)⁹pfu), and a phage without an inserted sequence was set as a control (blank phase), incubated at 4 ℃ for 30 min, washed twice with PBS 7.2, and the non-specifically bound phage was washed off, added with biotinylated-anti-M13 antibody, incubated at 4 ℃ for 30 min, and washed twice with PBS 7.2; adding streptavidin-PE and anti-mCD11c-APC, and incubating at 4 ℃ for 30 min; PBS 7.2 washing twice; and (5) judging the binding force condition by flow detection.

The flow assay results are shown in FIG. 1. Note that, CD11c⁺anti-M13-PE⁺The positive clone with the proportion higher than that of the control group has higher affinity with the mouse spleen DCAnd (4) harmony. As can be seen from fig. 1, the TY phage monoclonal showed significant affinity, which was 10.3% higher than the control group, and DK also showed some affinity.

In the present application, a whole cell screening method is used for screening polypeptides having affinity with mouse spleen DCs, which are derived from immature DCs having a relatively high antigen uptake ability. In the screening process, two rounds of the screening process are performed by incubating the DC in the spleen of the sorted mice with the phage, and two rounds of the screening process are performed by incubating the whole spleen cells with the amplified phage in the previous round. The reason is that after two rounds of screening, the phage having affinity with DC is enriched to a certain extent, considering that other cells are contained in the whole spleen cells, the phage is competitively combined with DC, monoclonal having stronger specificity and being capable of having affinity with DC is obtained, the final phage is enriched as shown by titer determination and statistical recovery rate results, and a plurality of phage monoclonal having better affinity with DC, especially TY phage monoclonal having significant affinity with DC, are preliminarily obtained through phage affinity DC experiment.

Example 2

Based on the TY peptides obtained by screening in example 1, the inventors synthesized affinity peptides TY peptides for DC by Fmoc solid phase synthesis. It should be noted that, since the affinity of the DC is verified by binding streptavidin with fluorescence to biotin, which indirectly reacts the affinity of the DC with the polypeptide, after the TY peptide is synthesized, a biotin is further linked to the carboxyl terminal of the TY peptide via the linker-GGG-. In order to facilitate the subsequent preparation of nano-carrier, the acetylated TY peptide and the antigen peptide OVA are simultaneously prepared_257-264. This example is briefly described below with respect to the preparation process.

(one) addition of the first amino acid

Weighing 0.37 g Rink resin, pouring into a synthesizer, adding DMF to swell the resin for about 30 min (if Wang resin is selected, deprotection is not needed); adding prepared deprotection solution (piperidine: DMF =1: 4) for deprotection for 2 times to remove the protecting group at the N end of the amino acid, and each time for 10 min;pumping the deprotection solution by using a vacuum pump, and washing the deprotection solution for 3 times by using DMF (dimethyl formamide) and 3 times by using DCM (DCM) for 2 min and 3 times by using DMF for 2 min; according to the formula: dosage = m_{Resin composition}Calculating the required dosage of amino acid, HCTU and DIEA by multiplying the molecular weight by equivalent (taking the equivalent as 4); weighing the first Fmoc-amino acid and HCTU of the corresponding carboxyl terminal of the polypeptide chain, fully dissolving the first Fmoc-amino acid and HCTU in a 4 mL EP tube by using 2 mL of DMF, adding the solution into a corresponding synthesizer, then adding 300 mu L of DIEA, and carrying out oscillation reaction on a speed-regulating oscillator for 2.5 hours; after washing, measuring a substitution value, draining liquid in a synthesizer, taking a small amount of resin, drying, weighing 1-1.5 mg of resin, adding 3 mL of deprotection solution, carrying out oscillation reaction for 10 min, then adding the resin into a cuvette, after zero setting, measuring the OD value of the resin at the wavelength of 290 nm by using an ultraviolet spectrophotometer, and calculating according to a formula: substitution value = OD₂₉₀Value/(1.65 Xm)_{Resin composition}) The substitution value is between 0.3 and 0.7, the reaction effect is optimal, if the substitution value is less than 0.3, the reaction between the amino acid and the resin is not good, and the amino acid needs to be added repeatedly for one time; because the first amino acid will not bind to all active sites of the resin, in order to avoid excessive byproducts, 3 mL of capping solution (acetic anhydride: pyridine solution =1: 1) is added to each synthesizer, and shaking reaction is performed on a shaking table for 2 times, each time for 20 min; and after the reaction is finished, pumping the end socket liquid in the synthesizer, and washing.

(II) addition of subsequent amino acids

After the first amino acid is successfully added, deprotection is carried out for 2 times, each time lasts for 10 min, and washing is carried out; after washing, picking the macroscopic resin by using a fine iron wire into a clean indene detection tube, adding an indene detection reagent, placing the indene detection reagent in a boiling water bath for reaction for 2 min, observing the color of the resin, and if the color of the resin is blue (but proline, serine and proline are generally reddish brown), indicating that the deprotection reaction is successful, otherwise, carrying out deprotection again; after the indene detection is successful, calculating and weighing the amount of the second amino acid and the HCTU to be added according to a formula, adding DMF (dimethyl formamide) into a 4 mL EP (ethylene-propylene-diene) tube to dissolve the second amino acid and the HCTU, adding the mixture into a synthesizer, adding a certain amount of DIEA, and carrying out oscillation reaction for 1 h; and (4) after washing, performing indene detection, and detecting whether the amino acid is successfully condensed with the carboxyl of the previous amino acid or not by the same operation, wherein if the resin is colorless and transparent and has no blue spots after the indene detection, the condensation reaction is successful, and otherwise, the amino acid needs to be added again.

The above procedure was repeated until the amino acid addition was complete.

It should be noted that, when preparing acetylated TY peptide, on the basis of the above synthesis steps, deprotection is performed first, then shaking reaction is performed on a shaking table for 2 times and 10 min/time, washing is performed, 3 mL of acetic anhydride is added into a synthesizer for 2 times, and shaking reaction is performed for 15 min/time, so that polypeptide acetylation can be achieved.

Cleavage of (tri) polypeptide chains

Cleavage is performed after successful (or acetylation) of the amino acid addition throughout the polypeptide chain. Carrying out deprotection before cutting, and carrying out shake reaction on a shaking table for 2 times and 10 min/time; washing, and washing with DCM for 3 times, each for 2 min; pumping out the liquid in the synthesizer, and performing indene detection to detect whether the protecting group at the N end of the last amino acid is completely removed; adding about 5mL of prepared cutting reagent into each synthesizer, and vibrating on an oscillator for reaction for at least 3 h; after the reaction, the reacted liquid was vacuum filtered into the round bottom flask by a vacuum pump in a fume hood, and an appropriate amount of DCM was added to wash the resin until the filtered liquid appeared colorless, and the washing liquid was also added into the round bottom flask.

Setting the temperature of a rotary evaporator to be 65 ℃, placing the round-bottom flask on the rotary evaporator for evaporating for 30 min to remove low-boiling organic reagents (TFA, DCM and the like), finally adding 1/3 volume of anhydrous ether into the round-bottom flask, and performing rotary evaporation for 5 times, wherein each time is about 15 min; adding 100 mL of ice ether into a round-bottom flask, observing that the liquid turns milky white, and precipitating on ice for 30 min to obtain a crude peptide precipitate preliminarily; abandoning the supernatant, resuspending the precipitate with glacial ethyl ether, transferring to a 15 mL centrifuge tube, marking, centrifuging at 2000 rpm for 2 min, abandoning the supernatant, resuspending the precipitate again, adding new glacial ethyl ether, and washing for 5 times to remove the organic reagent which is difficult to volatilize; placing the opening of the centrifuge tube for collecting the precipitate in a ventilation cabinet to volatilize overnight, completely volatilizing the diethyl ether, weighing, marking, and storing at-20 ℃ for later use.

(IV) high performance liquid chromatography purification and mass spectrometry detection

And (3) carrying out high performance liquid chromatography purification on the crude peptide cut in the step (three), wherein the specific chromatographic parameters are as follows: mobile phase solution, wherein the solution A is ultrapure water containing 0.1 per mill of trifluoroacetic acid, and the solution B is chromatographic grade acetonitrile; setting the wavelength to be 228 nm, gradually increasing the flow rate according to the sequence of 1 mL-3 mL-5 mL per minute in turn, then finally setting the flow rate to be 5 mL/min, and setting the elution gradient of 20% -60% acetonitrile; freeze-drying the collected sample in a freeze dryer, collecting in an EP tube, marking, sealing with a sealing film, and storing in a refrigerator at-20 deg.C.

During mass spectrum identification, a small amount of purified fine peptide (visible to the naked eye) after chromatographic purification is taken and placed in a 1.5mL EP tube, the molecular weight of the polypeptide is calculated by utilizing online software, a mass spectrum analyzer is adopted to analyze and determine the molecular weight of the polypeptide, and whether the molecular weight determined by an instrument is consistent with the molecular weight predicted by theory is compared so as to identify whether the polypeptide is successfully synthesized.

According to the above operation, the affinity peptide and the antigen peptide OVA of the biotin-labeled DC are finally successfully synthesized_257-264(Note here, OVA_257-264The fragments are OVA whole protein which can activate CD8 after being processed and enzymolyzed in vivo⁺The fragment sequence of the T cell needs to be used for an experiment during an in vitro stimulation experiment, but OVA holoprotein) and acetylated TY peptide need to be adopted when the in vivo effect is realized after the tumor is loaded on a mouse, an RP-HPLC purification system is utilized to successfully prepare the polypeptide, the collected corresponding peak products are analyzed and identified by mass spectrometry, the results are shown in figure 2, figure 3 and figure 4, the mass spectrometry result is consistent with the theoretical molecular weight of the polypeptide, and the successful synthesis of the polypeptide is illustrated.

Affinity peptide TY-biotin and antigen peptide OVA of biotin-labeled DC_257-264And the molecular weight and mass spectrometric detection of the acetylated TY peptide (TY-Ac) are shown in the following table:

it should be noted that, since in the present applicationThe sequence of the 12 peptide insert sequence of M13 phage in the NEB phage display peptide library technology used was XXX₁₂the-GGGS-pIII protein is characterized in that the C end of a 12 peptide sequence is connected with Gly to form an amido bond, therefore, Rink resin is selected in the synthesis process, and the synthesized polypeptide chain can form the amido bond at the C end. Based on the above, the synthesized biotin-labeled polypeptide is also modified at C-terminal, i.e. a terminal sequence GGGK is added^biotinAnd during subsequent acetylation, the prepared N-terminal acetylated TY peptide is used for the subsequent synthesis of the nano-carrier.

Example 3

In the actual use process of the screened TY peptide, the TY peptide is limited by the reasons of organism absorption, biological degradation and the like, so that the novel nano vaccine delivery system MSN-TY/OVA/CpG capable of targeting DC is prepared by taking nano material MSN (mesoporous silica nano particle) as a carrier. During preparation, firstly, acetylated TY peptide is covalently coupled to the surface of MSN through condensation reaction to synthesize MSN-TY, and then the mode antigen OVA and immune adjuvant CpG (CpG-ODN 1826) are loaded through electrostatic adsorption.

The specific process for preparing MSN-TY (connection of MSN-NH2 and COOH-peptide-Ac) is as follows:

dissolving a proper amount of acetylated TY peptide in 2 mL of DMF, adding 10-time equivalent of HoBt for mixed incubation, adding 10-time equivalent of DIC, uniformly mixing with the reactant, incubating at room temperature for 10 min, and activating carboxyl on the TY peptide;

according to the weight ratio of MSN-NH 2: measuring a proper amount of MSN (methyl methacrylate) in a clean 10 mL round-bottom flask according to the weight ratio of peptide) =1:1, redispersing MSN-NH2 in 2 mL DMF by using an ultrasonic cleaner, adding activated TY peptide into the MSN dispersed by ultrasonic, and carrying out magnetic stirring reaction for 24 hours at room temperature; the reaction product was washed 3 times with water to remove unbound polypeptide, and then dispersed in PBS buffer (pH = 7.2), and the above reactions were all performed with exclusion of light.

It should be noted that, in the preparation process, different load ratio experiments are respectively set for determining the load conditions of OVA and CpG on MSN, specifically:

dissolving OVA in PBS 7.2 to prepare a 1 mg/mL solution, adding prepared MSN-TY nanoparticles (MSN: OVA is 1.25:1, 2.5:1 and 5:1 in sequence) into a round-bottom flask according to different mass ratios, finally supplementing the volume to 2 mL, and placing the mixture on a magnetic stirrer at 4 ℃ to stir overnight; centrifuging at 4 deg.C and 10000 rpm for 30 min to remove unreacted OVA, collecting supernatant, determining protein content in the supernatant by BCA method, and calculating protein encapsulation efficiency;

；

on the other hand, different mass ratios of MSN and CpG (MSN: CpG is 2, 5, 10 and 20 in order) were stirred at 4 ℃ for 24 hours, unloaded CpG was removed by centrifugation, the supernatant was taken and subjected to Nanodrop 2000 to determine the CpG loading rate, and the CpG loading was analyzed by agarose gel electrophoresis.

When the prepared MSN-TY/OVA/CpG is characterized, the grafting ratio of the MSN and TY peptide is determined by Thermal Gravimetric Analysis (TGA) (when in Analysis, N is used₂Protection, heating rate is 10 deg.C min^-1) (ii) a An infrared spectrometer (FT/IR-4000) is adopted to represent organic groups (potassium bromide tablets, 4000-400 cm) on the surface of a sample^-1In-range scanning); observing the morphology of the nanoparticles by a transmission electron microscope (TEM, JEOL JEM-1200EX, Japan); the size distribution and Zeta potential of the different nanoparticles were observed using a particle size potential analyzer (Malvern Zetsizer Nano-ZS 90).

Before specifically preparing the MSN-TY/OVA/CpG nano-material, the inventors firstly carried out experimental analysis on the affinity of the affinity peptide and DC, and the specific experimental process is briefly described as follows.

(one) Induction of BMDC (bone marrow-derived dendritic cells)

Selecting 6-8 weeks SPF-grade C57BL/6J female mice, and sterilizing after killing (soaking in 75% alcohol for about 5 min); dissecting the mouse, collecting the hind leg bone, sterilizing with 75% ethanol, and washing with sterile PBS 7.2 for 2 times; cutting off the bone joint by using scissors to enable the two ends of the bone joint to be communicated, pouring a proper amount of culture medium (without serum) into a sterile culture dish, clamping the bone by using forceps with one hand, sucking the culture medium by using a 1 mL syringe with the other hand, inserting a syringe needle into the middle of the bone, pushing the syringe at a constant speed, observing red bone marrow at one side of a liquid, and repeating the washing process until the whole bone is white; filtering with a screen, collecting the cell suspension, centrifuging at 4 deg.C and 2000 rpm for 5 min, cracking, and washing with sterile PBS for 2 times (7.2 times); resuspending the cells in 5mL of RPMI1640 medium (containing 10% FBS and double antibody), evenly mixing, evenly dividing into 2 sterile culture dishes, supplementing the medium to 20 mL, adding recombinant mouse GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) cytokines, placing the culture dishes in a cell culture box for culture, and recording the day of induction as day 0; half of the culture medium is carefully extracted by using a pipette at the beginning of the 3 rd day, after centrifugation, the cells are added into a corresponding dish after being resuspended, two cytokines are added (if the cell density is too high, the dish separation should be considered), the liquid is changed once every day, and immature BMDC can be obtained at the 6 th day for subsequent experiments.

(II) detecting the affinity of the affinity peptide to DC

Immature BMDCs were harvested at day6 of induction and cell density was adjusted to 5X 10⁶Dividing each cell/mL into 1.5mL EP tubes on average, washing each tube with 100 mu L PBS for 1 time at 7.2 times, centrifuging, and discarding the supernatant; weighing biotin-labeled affinity peptide and control peptide by using a precision balance, diluting to 100 mu M by using PBS (phosphate buffer solution) 7.2, adding 100 mu L of diluted polypeptide into the centrifuge tube, and incubating with cells for 30 min at 4 ℃; washing with PBS 7.2 for 2 times, centrifuging at 4 deg.C and 3000 rpm for 5 min; adding 50 μ L PBS 7.2 containing 5% rat serum, placing at 4 deg.C, sealing for 10 min, adding anti-mCD11c-APC and streptavidin-PE, and incubating at 4 deg.C for 30 min; the affinity was checked by flow after adding 1 mL PBS 7.2 directly to wash 1, resuspending 200. mu.L PBS 7.2.

Although DC exists in various tissues in vivo, the content of the DC is low, the types of the DC are more, and the DC cannot meet the requirements of experimental research. In vitro induction from peripheral blood mononuclear cells (PMDCs) is often used for humans. For mice, bone marrow cells derived from mice, i.e., bone marrow-derived dcs (bmdcs), are most commonly used for induction. To further verify the affinity of the screened peptides to DC, the peptides were analyzed indirectly by flow-through using biotin-labeled polypeptides bound to the DC surface, followed by streptavidin (streptavidin) binding to biotin.

Figure 5 shows that only TY peptide was able to bind to DC and none of the remaining peptides did not bind to DC (control peptide GA peptide, DK peptide) (restricted to plate, blank control results without peptide not shown). Meanwhile, the affinity experiment for spleen-derived DC and whole spleen cells also shows similar results. Based on the result, a good foundation is laid for adopting TY peptide as the targeting peptide of the nano-carrier.

A brief introduction of the characterization of the MSN-TY, MSN-TY/OVA/CpG materials prepared is given below.

The results of characterizing MSN-TY by infrared spectroscopy are shown in FIG. 6 (FIG. 6A). The analysis shows that the MSN and the MSN-TY are 1630 cm^-1All show the same absorption peak and are attributed to H in the mesopores of MSN₂O, TY peptide was 1630 cm^-1The same peak appears in the region, which is attributed to the small amount of H remaining after the polypeptide is lyophilized₂And O. The infrared spectrogram of MSN-TY is at 1580 cm compared with MSN^-1And 1280 cm^-1The new absorption peaks appeared and are respectively attributed to the absorption peaks of amido bond I and amido bond II, which indicates that TY peptide is successfully coupled to the surface of MSN. Meanwhile, the grafting ratio of MSN-TY was calculated by thermogravimetric analysis (FIG. 6B). It can be seen from the figure that the weight loss before heating to 100 ℃ is due to residual moisture in the voids of the MSN, the weight losses of MSN and MSN-TY are 3.78% and 2.43%, respectively, and that the total weight loss of the two materials when heated to 800 ℃ is 14.79% and 24.43%, respectively, and the weight loss from 100 ℃ to 800 ℃ is due to decomposition of the organic functional groups. The grafting ratio can be represented by the formula (H1-L1)/(100-H1) = (H2-W-L1)/(100-H2). The final calculation gave a grafting yield result of 12.24%.

In the process of preparing the MSN-TY/OVA/CpG material, OVA is taken as a mode antigen, CpG is taken as an immunologic adjuvant, and the OVA and the CpG are loaded into a mesoporous pore channel of MSN or MSN-TY through electrostatic adsorption. After the reaction, the supernatant was centrifuged to remove the unloaded OVA, and the BCA protein quantification kit was used to analyze the protein loading.

As can be seen from fig. 7, as the MSN-TY input increased, the OVA loading increased, but when MSN: the OVA (w/w) was 97.6% at 5, and then the load rate was substantially constant as the amount of MSN increased. The CpG loading was analyzed by agarose gel electrophoresis of the supernatant after centrifugation using the same procedure (FIG. 7B). As can be seen from the results, when MSN: CpG (w/w) is 20, CpG bands are not substantially observed, indicating successful CpG adsorption to MSN-TY.

Analysis of a Transmission Electron Microscope (TEM) image (FIG. 8A) of the related material shows that the MSN, MSN-TY/OVA/CpG particles are all approximately spherical in shape, regular in morphology, good in dispersibility, proper in particle size and about 120 nm in average particle size. And FIG. 8B shows that the MSN nanoparticles are uniformly dispersed without precipitation and aggregation. The scanning electron microscope image of MSN is shown in FIG. 8C, in which the MSN particles are regular in morphology and uniform in size. The potentials of the different nanocarriers were measured using a malvern nanoparticle size potential analyzer (fig. 8D). FIG. 8E shows the particle size distribution of different Nano-carriers measured by a particle size potential analyzer (Nano-S90). The average particle sizes of MSN and MSN-TY were 156.8 nm and 155.02 nm, respectively, slightly larger than the TEM particle size. The particle size of the nanoparticles in the aqueous solution state is provided in the former, the TEM picture shows the particle size of the dried nanoparticles, and the results measured by the two methods are basically consistent, which shows that the nanoparticles are successfully prepared and have no obvious deformation phenomenon in water.

Example 4

Based on example 3, the inventors carried out preliminary analysis on the activity of nanocarrier MSN-TY/OVA/CpG in vitro, and the related experiments are briefly described as follows.

(I) cytotoxicity assay of MSN-TY/OVA/CpG

BMDCs were harvested on day6 of induction and cell suspension density adjusted to 5X 10 in RPMI-1640 complete medium with 10% FBS⁵ cells/mL, 50000 cells/well in 96-well plates, 100. mu.L/well, 37 ℃, 5% CO₂Culturing for 2 h in an incubator;

experimental groups (5 duplicate wells per group) were as follows: zero setting hole: cell-free PBS group only; control group: PBS + cells; experimental groups: OVA group, MSN/OVA/CpG group, MSN-TY/OVA/CpG group, each well added with 100 u L serum-free medium diluted drug, according to the concentration of OVA, the following in turn: 12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, and cultured in an incubator for 48 hours.

Adding 20 mu L of MTT solution into each hole 4h before finishing, continuously incubating for 4h, terminating the culture, discarding the old culture solution, adding 150 mu L of DMSO into each hole, and shaking at room temperature for 10 min to fully dissolve the crystals; the absorbance of each well was measured at 490 nm and the results recorded. And drawing a cell growth curve by taking the absorbance value as a vertical coordinate and different concentrations of the medicine as a horizontal coordinate.

The in vivo and in vitro toxicity of the nano-carrier is an important factor for restricting the application of the carrier in the biological field. Therefore, we analyzed the cytotoxic effect of the constructed vector pair using the MTT assay. By using model cells of BMDC, when different nano-carriers and BMDC cells are incubated for 48 h, and the OVA content in the carriers is changed from 12.5 mug/mL-100 mug/mL, along with the increase of the carrier concentration, compared with a control group without the carriers, the OD value has no obvious change (shown by 9A), and the result shows that the MSN-TY/OVA/CpG nano-carriers have no obvious influence on the growth of the cells, have no toxic effect, and can be used for subsequent in vivo and in vitro experiments.

(II) cellular uptake assay of MSN-TY/OVA/CpG

In the flow detection, the following experimental methods are specifically referred to: after labeling OVA protein with FITC (fluorescein isothiocyanate) first with reference to the FITC-NHS instructions from Sigma, the experiment was performed by reference to the following procedure:

taking spleen cells of C57BL/6J mouse, grinding, cracking to red, making into single cell suspension, spreading on 96-well plate (U-shaped bottom), adjusting cell density to 1 × 10⁷cells/mL, 200 uL per well; respectively with OVA_FITC/CPG、MSN/OVA_FITC(CPG) and MSN-TY/OVA_FITC/CpG (containing OVA)_FITC20 ug/ml) of carbon dioxide at 37 deg.C and 5% CO₂Incubating for 4 hours in an incubator; PBS 7.2 Wash twice, 4 deg.CCentrifuging at 3000 rpm for 5 min; then adding anti-mCD11c-APC, incubating at 4 ℃ for 30 min, and washing with PBS 7.2 once; resuspend cells with 200 μ L fixative (diluted to 1 ×) and fix for 30 min at room temperature; then 200 μ L of the resulting solution was washed with membrane-breaking agent (diluted to 1X with sterile water) 2 times, and centrifuged at 4 ℃ and 3000 rpm for 5 min; PBS 7.2 resuspend the cells, wash once, and detect on the flow machine.

When the laser confocal detection is adopted to detect the cellular uptake of MSN-TY/OVA/CpG, the following experimental methods are referenced:

collecting BMDCs inducing immature at day6, adjusting cell density to 1 × 10 with serum-free medium⁶2 mL of seed/mL, per well, in 6-well plates; adding OVA_FITC/CpG、MSN/OVA_FITC/CpG、MSN-TY/OVA_FITCCpG at 37 ℃ with 5% CO₂Incubate for 4h in incubator (groups OVA)_FITCThe final concentration is 20 mug/mL); collecting cells, transferring to 1.5mL EP tube, washing twice with PBS 7.2, centrifuging at 4 deg.C and 4000 rpm for 5 min; diluting Lysotracker Red DND-99 with serum-free medium to make the final concentration of working solution be 50 nM, preheating in 37 deg.C water bath, adding 1 mL of the above working solution into each tube, and placing at 37 deg.C with 5% CO₂Incubating the incubator and the cells for 1 h; PBS 7.2 washing twice; adding Hoechst 33342 (preheated at 37 ℃) with the final concentration of 2 mug/mL, putting the mixture into an incubator for cell co-incubation for 40 min, and washing the cells twice by PBS; fixing 4% paraformaldehyde at room temperature for 15 min, washing with PBS twice, finally resuspending 10 μ L, adding appropriate amount of anti-fluorescence quencher, dripping onto glass slide, covering with cover glass, sealing, and observing under confocal microscope.

Because of the efficient antigen transport and the ability of the antigen to be taken up by the DC, it is an important step in the activation of immune responses in vivo in tumor vaccine therapy. By means of covalent coupling of FITC to OVA, through constructing a fluorescent nano-carrier, spleen DC is subjected to different nano-carrier OVA by using a flow detection method_FITC/CpG、MSN/OVA_FITCCpG and MSN-TY/OVA_FITCThe results of the detection and analysis of the CpG uptake ability are shown in FIGS. 9B and 9C. The analysis can show that the MSN-TY/OVA_FITCThe fluorescence intensity of the/CpG treated group was strongest and significantly higher than that of the remaining experimental groups, whereas MS wasN/OVA_FITCthe/CpG treated group was higher than free OVA_FITCthe/CpG group, this result indicates, with free OVA_FITCNo targeting of MSN/OVA compared to CpG_FITCthe/CpG nanocarriers also exhibit certain advantages, namely, nanoparticle-mediated transport allowing uptake of OVA by DC_FITCEnhanced capacity, based on which MSN-TY/OVA_FITCTY peptide in the CpG group plays a targeting role, is prevented from being taken up by macrophages, and can effectively improve the taking up of DC to the nano-carrier.

For free OVA_FITCAnd confocal microscopy observations of different nanocarriers after incubation with immature BMDCs are shown in figure 9D. In the color image, blue is the nucleus, red is the lysosome, and green is the nano-carrier, and it can be seen that OVA_FITCthe/CpG treated group showed only weak green fluorescence, while the nanocarrier group showed strong green fluorescence, in which MSN-TY/OVA_FITCThe fluorescence of the/CpG group is strongest, which indicates that the nano-carrier can be efficiently taken up by BMDC, and OVA can be observed_FITCColocalization around lysosomes occurred (yellow fluorescence appeared by the superposition of green and red fluorescence), indicating that the nanocarriers entered the lysosomes after uptake by BMDC.

The comprehensive results can be identified as follows: the MSN-TY/OVA/CpG nano-carrier can effectively transport antigen, and is recognized and taken up and processed by DC, which is particularly key to activating the antigen specific immune response of the organism.

(III) Effect of MSN-TY/OVA/CpG on DC maturation

Firstly, flow detection is carried out on the change condition of a marker on the BMDC surface, and the specific operation references are as follows:

BMDCs from day6 of induction were plated in 6-well plates at 2X 10 per well⁶Adding different nano-carriers, OVA/CpG, MSN/OVA/CpG and MSN-TY/OVA/CpG into each cell, adjusting the concentration of OVA loaded by the carriers to be 1 mg/mL by PBS 7.2, adding the corresponding carriers to each hole to contain 100 mu g of OVA and 30 mu g of CpG, simultaneously setting a blank control of adding a corresponding amount of PBS 7.2 without a loading body, and adding 5% CO at 37 ℃ under the condition of 5 percent CO₂Culturing for 24 hours in an incubator; collecting supernatant, freezing at-80 deg.CELISA to detect the secretion of cytokines (TNF-alpha and IL-12p 70); collecting the cells, adding antibodies anti-mCD40-FITC and anti-mCD86-FITC into a correspondingly marked centrifuge tube, respectively adding anti-mCD11c-APC, setting a homotypic control tube and a single positive tube of CD40 and CD86, and incubating for 30 min at 4 ℃ in the dark; and centrifuging, washing, then resuspending by 200 mu L, and carrying out flow detection on the change conditions of the DC surface co-stimulatory molecules CD86 and CD 40.

When ELISA is used for detecting the secretion of cytokines (TNF-alpha and IL-12p 70), the specific operation is as follows:

diluting 10 multiplied by coating liquid into 1 multiplied by coating liquid which is working liquid, diluting capture antibody, namely Anti-mouse TNF-alpha or Anti-mouse IL-12p70, to the required concentration according to the ratio of 1:1000, adding the diluted capture antibody into a 96-well plate by a discharging gun, placing the plate in a closed wet box, and coating the plate overnight at 4 ℃; the next day, discarding the coating solution, drying on clean filter paper, adding 300 muL/well PBST buffer solution containing 0.05% Tween-20, staying for 1 min, discarding the liquid in the pore space, drying, and repeating for 3-5 times; adding 200 mu L of 1 xAssay Diluen buffer solution into each hole, and sealing for 1h at room temperature; washing 1 time with PBST (PBS 7.2 buffer containing 0.05% Tween-20); diluting the Mouse TNF-alpha or Mouse IL-12p70 standard sample and the supernatant sample of the culture medium to be detected with a 1 xAssay Diluen buffer solution gradient, covering a sealing plate membrane for incubation for 2 h at room temperature, wherein the sample is 100 muL/well; washing for 3-5 times; diluting a detection antibody (Anti-mouse TNF-alpha-biotin or Anti-mouse IL12p70-biotin, diluted 1: 1000) with 1 × Assay Diluen, 100 μ L/well, and incubating at room temperature for 1 h; washing for 3-5 times; diluting Avidin-HRP to a working solution concentration of 100 muL/well by using 1 xAssay Diluen according to a ratio of 1:500, incubating for 30 min at room temperature, and keeping out of the sun; washing for 5-7 times; adding a substrate 1 XTMB color development solution of HRP, incubating for 15 min at the temperature of 37 ℃ in a dark place, wherein the color development can achieve a good effect within 10-20 min generally; 50 μ L of 1 mM H was added rapidly to each well with a row gun₃PO₄Stopping the color reaction by using the solution; and (3) detection: and (3) reading an absorbance value (OD value) at 450 nm by using a microplate reader within 15 min, making a standard curve according to the reading of TNF-alpha and IL-12p70 standard substances, and calculating the concentration of TNF-alpha and IL-12p70 in the detection sample.

DCs are professional antigen presenting cells that can activate innate and adaptive immune responses. Recognition and capture of tumor antigens by immature DCs is the first step in mediating T cell immune responses. Mature DCs up-regulate costimulatory molecules (CD 40 and CD 86), which are important indicators for the activation of T cell-mediated immune responses.

The results of experiments on whether MSN-TY/OVA/CpG could activate DCs, promote DC maturation and up-regulate co-stimulatory molecules are shown in FIG. 10. Analysis showed that the expression of CD86 was significantly increased in the MSN-TY/OVA/CpG treated group (FIG. 10A and FIG. 10C), the up-regulation of CD80 promoted the immune response of primary and secondary T cells, and CD40 was a co-stimulatory molecule important for both innate and adaptive immunity. Compared with the PBS group, the expression level of CD40 in the MSN-TY/OVA/CpG treated group was increased by 58.7%, the expression level in the OVA/CpG treated group was increased by 13.8%, and the expression level in the MSN/OVA/CpG treated group was increased by 50.4% (FIG. 10B and FIG. 10D). Various cytokines secreted by mature DCs are also of paramount importance in anti-tumor immune responses.

To further verify the role of MSN-TY/OVA/CpG in antigen presentation, the secretion test results of ELISA on TNF- α (FIG. 10E) and IL-12p70 (FIG. 10F) showed that the PBS group as the control group, TNF- α and IL-12p70 were significantly higher in the MSN-TY/OVA/CpG treated groups than in the other groups, wherein the secretion of both the OVA/CpG and MSN/OVA/CpG treated groups was also somewhat higher, and the nanocarrier group was superior to the free OVA group, indicating that antigen presentation to DC could indeed promote DC maturation and activation to some extent, and TY targeting peptide coupling could improve the antigen transport efficiency of the nanocarrier system, which would be beneficial for promoting T cell proliferation and activation of subsequent immune response.

(IV) antigen Cross-presentation of MSN-TY/OVA/CpG-Targeted DCs

The specific experimental process is as follows:

(1) nano-carrier with different immature BMDC load

Specifically, the method comprises the following steps: immature BMDCs were plated in 96-well plates (Round button), 2X 10⁵ Adding OVA containing 50 mug/mL and OVA containing 500 mug/mL respectively to the cells/wellOVA/CpG, MSN/OVA/CpG and MSN-TY/OVA/CpG of nM CpG, 4 holes per group, BMDC group without peptide, 5% CO at 37 deg.C₂Incubating for 4h in an incubator; after the incubation, the cells were centrifuged at 2000 rpm for 5 min at 4 ℃ and washed 2 times in serum-free RPMI1640, and the cells were counted and adjusted to a density of 1X 10⁵one/mL for use.

(2) BMDC and CD8⁺T cell co-culture

With reference to the previous procedure, CD8+ T cells from OT-I mice were sorted using the CD8+ T sorting kit, and then made 1X 10 by labeling CD8+ T cells with CFSE⁶Suspension per mL for use. Marked CD8⁺Laying the T cells in a 96-well plate with a round bottom, wherein each well is 100 muL; after 30 min, the nanocarrier-loaded BMDC or untreated BMDC (control) were added to a 96-well plate at 100 μ L per well and mixed with CD8⁺T cells were co-cultured for 72 h, and final BMDCs were co-cultured with CD8⁺The ratio of T cells is 1: 10; after the co-culture is finished, collecting supernatant, freezing and storing at-80 ℃, and detecting the secretion amount of IFN-gamma by ELISA the next day; a portion of the cells are flow tested for proliferation of T cells; the other part of the cells (5X 10)^2-8One) was collected into a 1.5mL EP tube, 500. mu.L of RNA lysate was added, and the cells were repeatedly blown up with a gun until all cells were lysed, and stored at-80 ℃. The expression level of perforin and Grz B at mRNA level was determined by qRT-PCR.

Flow detection of CD8 in coculture system⁺In the case of proliferation of T cells, the detection method is:

after the co-incubation was completed, BMDC and CD8 were collected⁺T cells were centrifuged in 1.5mL EP tubes and washed once with PBS 7.2;

adding the antibodies anti-mCD3-PerCP-eflour710 and anti-mCD8 alpha-APC diluted by PBS 7.2, placing the cells on ice for incubation for 30 min after resuspension; and washing the cells for 2 times by PBS 7.2, and carrying out flow-type detection on the proliferation condition of the T cells after 200 microliter of PBS 7.2 is resuspended.

RNA extraction, cDNA reverse transcription and fluorescent quantitative PCR (beta-actin is used as an internal reference gene in detection) are carried out according to the conventional operation of the prior art, and are not described any more.

To verify whether DC uptake of antigen was further activatedEffective antigen cross-presentation and elicited T cell immune responses, the inventors used a classical in vitro model, namely antigen OVA-loaded DC cells and sorted OT-I mouse CD8⁺T cells were co-incubated to verify the effect of TY peptide targeting DC on the cross-presentation ability of OVA antigens.

The flow-type detection result of the proliferation of the nameive T cells is shown in FIG. 11A, and it can be seen that compared with the MSN/OVA/CpG group without targeting peptide TY, the fluorescence signal intensity of the CFSE-labeled T cells is gradually reduced in the MSN-TY/OVA/CpG-treated group, which indicates that the T cells are proliferated, the antigen is effectively transported, and the proliferation capacity of the antigen-stimulated T cells is further improved. Free OVA antigen, which may also be taken up directly by DCs, stimulates T cell proliferation to some extent. Whereas, in the PBS group, there was no stimulation of antigen by DCs, which could not be activated and thus could not stimulate T cell proliferation.

To further investigate the function of T cells after proliferation, we examined the IFN-. gamma.content of the supernatant in the co-incubation system of DC and T cells (FIG. 11B). The secretion of IFN-gamma in MSN-TY/OVA/CpG group is obviously higher than that in the other three groups, and the secretion is combined with CD8⁺The results of T cell proliferation were consistent. Therefore, MSN-TY/OVA/CpG can not only cause the proliferation of T cells but also activate the functions of the T cells and improve the secretion of IFN-gamma.

Meanwhile, the expression conditions of T cell surface killer molecules perforin and Grz B are detected by using a fluorescent quantitative PCR technology. The results show that the expression levels of the MSN/OVA/CpG group without the targeted peptide modification are not significantly different from those of the OVA/CpG group but are higher than those of the control group, while the expression levels of the MSN-TY/OVA/CpG treated group are obviously increased (shown in FIG. 11C and FIG. 11D).

After the immature DC captures OVA antigen, the antigen is processed and degraded into small peptide fragments, the DC is also activated, and a marker on the surface of the DC can be up-regulated and secrete cytokines to be further presented to T cells, so that antigen-specific T cell-mediated or antibody-mediated immune response is generated. Combining the above results, it can be seen that: the nano-carrier can efficiently mediate antigen transportation, and the phagocytosis efficiency of the targeting peptide TY-modified nano-carrier is further improved. And MSN-TY/OVA/CpG can activate DC, promote the maturation of DC, further enhance the antigen cross presentation effect, stimulate the proliferation of T cells, activate the functions of T cells, promote the release of cell factors TNF-alpha and IL-12p70, target and transport exogenous antigen to DC, cause effective antigen processing presentation effect, effectively stimulate the proliferation of T cells and the secretion of IFN-gamma, and improve the relative expression of T cell killing molecules perforin and Grz B on mRNA level.

Example 5

Based on the experiments in example 4, the inventors further carried out experimental analysis on the in vivo immune activity of MSN-TY/OVA/CpG. The relevant experiments are briefly described below.

(1) Immune animal model test

Dividing female C57BL/6J mice of 6-8 weeks into four groups (Control group, OVA/CpG group, MSN/OVA/CpG group, MSN-TY/OVA/CpG group) and 5 mice per group; injecting OVA/CpG and different nano-carriers into roots of the tail base at multiple subcutaneous points, wherein the injection dose of each group is calculated according to OVA and CpG ODN1826 loaded by the carriers, each mouse has 100 mu g of OVA and 30 mu g of CpG ODN1826, and a control group is simultaneously injected with the same dose of PBS 7.2 dissolved CpG ODN 1826; the immunization is carried out once every 7 days, and the administration is carried out three times; on the fifth day after the last immunization of the mice, the mice were sacrificed (overall scheme is shown in fig. 12A). Taking the spleen, and preparing the single cell suspension by the operation.

Adjusting the density of partial cells to 2X 10⁶After each mL, 1 mL of the antigen peptide is inoculated into a 24-well plate, the centrifugal tube is shaken up and down continuously during the process to prevent cell precipitation, and finally the antigen peptide OVA is added_257-274(final concentration 10. mu.g/mL), 1. mu.L of Protein Transport inhibitor (1: 1000 dilution) was added to each well, mixed well, and 5% CO was added at 37 ℃₂Culturing for 6h in an incubator, and detecting CD8 alpha by intracellular factor staining⁺The expression level of IFN-gamma in T cells;

adjusting the density of partial cells to 5 × 10⁶After each mL, 5mL of cells were inoculated in 6-well plates using a pipette, and antigen peptide OVA was added to both the experimental and control groups_257-274(final concentration 10. mu.g/mL), 5% CO at 37 ℃₂Culturing in an incubator for 5 days; collecting the supernatantThe cells were stored at-80 ℃ and assayed for IFN-. gamma.secretion by ELISA. And adding RNA lysate into the induced cells, blowing, beating and uniformly mixing, storing at the temperature of minus 80 ℃, extracting total RNA, and detecting the relative expression condition of perforin and Grz B by utilizing qRT-PCR after reverse transcription.

The experimental results showed that the MSN-TY/OVA/CpG treated group showed a stronger immune response, IFN-gamma, than the other groups⁺ CD8⁺The proportion of cells was 1.89%, but the MSN/OVA/CpG group IFN-. gamma.⁺ CD8⁺The proportion of cells was not as high as the OVA/CpG group (FIG. 12B). The ELISA test results were consistent with those of intracellular factor staining (FIG. 12C), and it can be seen that IFN-. gamma.was secreted in the MSN-TY/OVA/CpG-treated group in a significantly higher amount than in the remaining treated groups. And simultaneously, qRT-PCR detects the relative expression of CTL killing surface molecules peforin and GrzB on the mRAN level, and the expression quantity of the two is obviously multiplied compared with that of a control group. These results all indicate that MSN-TY/OVA/CpG enhances antigen-specific CTL responses.

(2) MSN-TY/OVA/CpG antitumor assay on B16-OVA tumor-bearing model

When carrying out the related tumor-bearing test, firstly establishing a B16-OVA melanoma transplantation tumor model, specifically:

collecting logarithmic phase B16-OVA cells, digesting with 0.25% pancreatin, blowing uniformly, adjusting cell density to 1 × 10 in RPMI-1640 culture medium⁶Placing the seeds/mL on ice; selecting 6-8 weeks of C57BL/6J female mice, extracting B16-OVA single cell suspension with a syringe, injecting the suspension into the right side and the lower back of the female mice subcutaneously, each 200 μ L, namely 2 × 10⁵Recording the day of each tumor on day 0;

on day 3 of tumor bearing, mice were randomly divided into 4 groups (Control group, OVA/CpG group, MSN/OVA/CpG group, MSN-TY/OVA/CpG group), each group consisting of 5 mice; injecting OVA/CpG and different nano-carriers into roots of the tail base at multiple subcutaneous points, wherein the injection dose of each group is calculated according to OVA and CpG ODN1826 loaded by the carriers, each mouse has 100 mu g of OVA and 30 mu g of CpG ODN1826, meanwhile, a control group is set to inject PBS 7.2 dissolved CpG ODN1826 with the same dose, and the immunization is carried out once every 7 days for three times; the length, width and height of the mouse tumor were measured every other day starting from the seventh day after tumor loading according to the formula: volume =1/2 (a × b × c) tumor volume was calculated and growth curves of the transplanted tumors were plotted (overall flow is shown in fig. 13A). And simultaneously measuring the body weight of the mouse and drawing a body weight change curve.

Detection of tumor infiltration CD3⁺CD8⁺For T cell ratios, the following procedure was referenced:

on the fifth day after the last administration, mice were sacrificed, then 5 tumor tissues of the same group were ground and crushed by ground glass slides (one centrifuge tube for each mouse, one pair of glass slides), the glass slides and the dish were washed by PBS 7.2, and the single cell suspension was screened to obtain a single cell suspension; centrifuging at 4 deg.C and 3000 rpm for 5 min to obtain cell precipitate, resuspending with 1 mL, transferring into 1.5mL EP tube, centrifuging, and discarding supernatant; diluting the following antibodies anti-mCD45-FITC, anti-mCD 3-eflor 710 and anti-mCD8 alpha-APC with PBS 7.2, adding the diluted antibodies into corresponding centrifuge tubes, resuspending cells, arranging a single positive tube with CD45, CD3 and CD8 alpha for machine-time regulation compensation, and incubating for 30 min at 4 ℃; washing with PBS 7.2, centrifuging at 4 deg.C and 3000 rpm for 5 min, resuspending with 200 μ L PBS 7.2, filtering, and testing on a computer.

ex vivoWhen the anti-tumor effect of MSN-TY/OVA/CpG is detected through experiments, the draining lymph nodes and spleen of mice are respectively taken to prepare single cell suspensions, and the intracellular factor staining is used for respectively detecting the CD8 alpha secreting IFN-gamma, perforin and Grz B in the draining lymph nodes by referring to the operation method⁺Proportion of T cells and IFN-gamma secreting CD8 alpha in spleen⁺The proportion of T cells; detecting the secretion of IFN-gamma in the culture supernatant of the spleen and the draining lymph nodes by ELISA; the relative expression of mRNA of perforin and Grz B was detected by qRT-PCR.

As can be seen from the growth curve of the mice (shown in FIG. 13B), the weight change of the mice gradually increases during the administration period, the growth condition of the mice is good, and the nano-carriers are nontoxic and have good biological safety. As shown in FIG. 13C, MSN-TY/OVA/CpG group had no significant tumor growth, while MSN/OVA/CpG and OVA/CpG groups also inhibited tumor growth to some extent, and MSN/OVA/CpG group (106.97 mm 3) showed better tumor inhibition than free OVA/CpG group (197.68 mm 3). Since OVA loading into MSN prolongs the circulation time of the antigen in vivo, it can be efficiently taken up by Antigen Presenting Cells (APC). On the fifth day of the last dose, mice were sacrificed and a photograph of the tumors of the mice was taken (fig. 13D). The above results all indicate that MSN-TY/OVA/CpG can effectively inhibit tumor growth.

For mouse tumor part CD3⁺ CD8⁺T cell infiltration analysis showed that MSN-TY/OVA/CpG treated group CD3 as shown in FIG. 14A⁺ CD8⁺The proportion of T cells was 32%, which was significantly higher than the rest of the treatment groups, suggesting that MSN-TY/OVA/CpG can significantly elicit antigen-specific CD3⁺CD8⁺T cells infiltrate into the tumor. Antigen peptide OVA for part of draining lymph node of immunized mouse_257-264And (3) stimulating in vitro for 6h, and carrying out flow detection on changes of intracellular IFN-gamma, perforin and GrzB. As shown in FIG. 14B, FIG. 15C, FIG. 15D, the frequencies of IFN-. gamma. + CD8+ T cells, perforin + CD8+ T cells and GrzB + CD8+ T cells were significantly higher in the MSN/OVA/CpG and OVA/CpG groups in the MSN-TY/OVA/CpG groups, and the spleen IFN-. gamma. + CD8+ T cells were also stimulated to increase in the MSN-TY/OVA/CpG groups (FIG. 15E). Spleen cell antigen peptide OVA of tumor-bearing mice_257-264The MSN-TY/OVA/CpG treated group significantly promoted IFN- γ secretion in the spleen (FIG. 16F) and lymph node (FIG. 16G) supernatants for 5 days of in vitro restimulation. And the relative expression levels of spleen cells, peforin (FIG. 16H) and GrzB (FIG. 16I) were increased. This is consistent with the results in an immune animal model. Experimental results show that the MSN-TY/OVA/CpG nano-carrier can effectively transport antigen in vivo in a targeted manner, can trigger spleen and drainage lymph node parts to generate antigen-specific CD8+ T cell immunoreaction, and enhances the anti-tumor effect.

The application utilizes MSN load mode antigen OVA and TLR9 ligand CpG, and the outer surface is connected with DC-targeted affinity peptide TY to prepare the MSN-TY/OVA/CpG nano-carrier. Immune animal experiments and B16-OVA tumor-bearing mouse model experiments both prove that the MSN-TY/OVA/CpG nano-carrier can carry antigen target to DC with high efficiency, trigger antigen-specific CTL immune response and remarkably stimulate CD8⁺Secretion of IFN-. gamma.by T cells and phase of cell-killing molecules perforin and GrzB at mRNA levelFor the expression amount.

In a tumor treatment model, the MSN-TY/OVA/CpG nano-carrier achieves better effect of inhibiting tumor growth. Increased CD8 in dLN⁺Intracellular IFN-. gamma.Perforin and GrzB levels in T cells, and spleen CD8⁺The release amount of IFN-gamma in the T cell is in the cell and the extracellular release amount of IFN-gamma in the T cell, and correspondingly, the relative expression amount of the cell killing molecules perforin and GrzB is also improved. The above results indicate that MSN-TY can be used as an effective antigen delivery vehicle, enhancing the antitumor effect.

Sequence listing

<110> Zhengzhou university

<120> a dendritic cell-targeting affinity peptide TY peptide and uses thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Synthesis

<400> 1

Thr Ile Thr His Phe Gln Phe Gly Pro Thr Val Tyr

1 5 10

Claims (7)

1. An affinity peptide TY peptide of a targeted dendritic cell is characterized in that the TY peptide is 12 peptide, the amino acid sequence of the TY peptide is shown as SEQ ID NO.1, and the specific amino acid sequence is as follows: Thr-lle-Thr-His-Phe-Gln-Phe-Gly-Pro-Thr-Val-Tyr, i.e., T-I-T-H-F-Q-F-G-P-T-V-Y, TITHFQFGPTVY.

2. Use of a dendritic cell targeting affinity peptide TY peptide according to claim 1 for the preparation of an anti-tumor vaccine.

3. The method for preparing a dendritic cell targeting affinity peptide TY of claim 1, wherein the method is performed by Fmoc solid phase synthesis.

4. Nanocarrier MSN-TY prepared from a dendritic cell targeting affinity peptide TY peptide according to claim 1, wherein the TY peptide is acetylated and covalently coupled to the MSN surface by:

activating carboxyl on acetylated TY peptide by utilizing HoBt and DIC, and then reacting with amination modified MSN to prepare the compound.

5. The use of the nanocarrier MSN-TY of claim 4 for the preparation of an anti-tumor vaccine.

6. The nano-carrier MSN-TY/OVA/CpG prepared by using the dendritic cell targeting affinity peptide TY peptide of claim 1 is characterized in that TY peptide is acetylated and then covalently coupled to an aminated and modified MSN surface to obtain MSN-TY, and then a mode antigen OVA protein and a TLR9 receptor agonist CpG are respectively adsorbed to the MSN-TY surface by an electrostatic adsorption method, so that the nano-carrier MSN-TY/OVA/CpG capable of specifically targeting dendritic cells is finally prepared.

7. The use of the nanocarrier MSN-TY/OVA/CpG of claim 6 in the preparation of an anti-tumor vaccine.

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